There are three types of influenza viruses: INFA, INFB, and influenza C (“INFC”). The most virulent influenza virus is influenza virus A, which can infect humans, birds, pigs, horses, seals, whales, and other animals. Influenza viruses that use wild birds as natural hosts are referred to as avian influenza viruses and influenza viruses that use humans as natural hosts are referred to as human influenza viruses. Domesticated birds, such as turkeys and chickens, have developed fatal illnesses from avian influenza virus, as have other animals and humans that have become infected with avian influenza virus through contact with infected domesticated birds. Domesticated birds may become infected with avian influenza virus through direct contact with infected wild birds, other infected animals, contact with surfaces harboring viruses, or contaminated food or water. Thus far, avian influenza viruses that have crossed species and infected humans are responsible for recent human influenza pandemics. The influenza B and influenza C viruses, both of which normally only infect humans, are less virulent than influenza A. While influenza B has been responsible for localized epidemics of influenza, it has not been the cause of any widespread influenza pandemics. The least virulent influenza C virus has never led to any widespread human influenza epidemics.
The influenza virus is an enveloped virus with a genome containing eight single-stranded negative sense RNA segments. The viral envelope has a host-derived lipid bilayer with two major surface viral glycoproteins: hemagglutinin (“HA”) and neuraminidase (“NA”), which are the proteins responsible for viral attachment. Within the envelope, matrix protein M1 and nucleoprotein (“NP”) protect the viral RNA. The A, B, and C type designation of the influenza virus is based upon the antigenic features of the M1 matrix protein and NP. The eight RNA segments encode at least 10 viral proteins: segments 1, 2, and 3 encode three viral polymerase proteins; segment 4 encodes HA; segment 5 encodes NP; segment 6 encodes NA; segment 7 encodes the M1 and M2 matrix proteins, the former which has ion channel activity and is embedded in the viral envelope; and segment 8 encodes the nonstructural proteins NS1 and NS2, the former which blocks the hosts antiviral response and the latter which participates in the assembly of virus particles.
INFA viruses are identified by the subtype of the HA and NA proteins on the surface of the virus. INFA viruses have 16 different HA subtypes and 9 different NA subtypes, all of which may exist on the surface of the virus in many different combinations; thus, an H5N1 virus has an HAS protein and an NA1 protein on its surface. All subtypes of INFA viruses are found in birds. The INFA subtypes commonly found in humans are the H1, H2, and H3 subtypes (H2 subtypes are currently not circulating) with the H5, H7, and H9 subtypes also having been known to infect humans. Among the INFA viruses found in birds, the H5 and H7 subtypes are the most virulent; strains of the H5 and H7 subtypes are further classified as either low pathogenic avian influenza (“LPAI”) or high pathogenic avian influenza (“HPAI”). HPAI are characterized by HAs that are highly susceptible to cleavage by numerous cellular proteases, which are widespread in cell compartments and organ systems; by contrast, LPAIs require specific active extra-cellular proteases, such as trypsin, which are restricted to the lumen of the respiratory and intestinal sites, for cleavage. Of domesticated birds infected with HPAI H5 or H7 viruses, 90% to 100% of the birds will die. Because LPAI H5 and H7 viruses can evolve into HPAI H5 and H7 viruses, respectively, outbreaks of LPAI H5 and H7 viruses in domesticated bird populations must be closely monitored. Subtypes of avian influenza virus circulating among animals and humans include the H7N7 and H3N8 viruses, which cause illness in horses, and the H1N1, H1N2, and H3N2 viruses, which are in general circulation among humans. INFB and INFC viruses are not classified according to subtype.
Since 1997, INFA viruses previously exclusive to infection in birds have been infecting humans with fatal outcomes. Confirmed outbreaks of avian influenza virus with some resultant human deaths have been reported in 1997 (H5N1 in Hong Kong), 1999 (H9N2 in China and Hong Kong), 2002 (H7N2 in Virginia, USA), 2003 (H5N1 in China and Hong Kong; H7N7 in the Netherlands; H9N2 in Hong Kong; H7N2 in New York, USA); 2004 (H5N1 in Thailand and Vietnam; H7N3 in Canada); and 2005 (H5N1 in Thailand and Vietnam).
Because avian IFNA viruses are carried globally via migratory birds and the virus is known to change rapidly as a result of antigenic drift and shirt and genetic drift, methods used for surveillance of avian influenza virus must have sufficient specificity to allow detection of antigenically and genetically diverse influenza strains.
Traditional methods to detect avian INFA include plaque assays, such as Culture Enhanced Enzyme Linked Immunosorbent Assay (“CE-ELISA”) and virus isolation in embryonated chicken eggs. Hemagglutinin and neuraminidase subtyping of the virus is carried out after detection by serological methods. While the traditional methods have been shown to be sufficiently sensitive, the processes are time-consuming; for example, virus isolation in embryonated eggs takes from one to two weeks to obtain results.
To overcome the time and cost disadvantages of the traditional methods of detecting avian INFA, diagnostic methods using the technique known as real time reverse transcriptase polymerase chain reaction (“real time RT-PCR”), also called kinetic RT-PCR (“kRT-PCR”), have been developed. Such assays detect INFA and INFB using sequences derived from the INFA and INFB matrix and nucleoprotein genes. Stone et al., J. VIROL. METH. 117:103-112 (2003); Smith et al., J. CLIN. VIR. 28:51-58 (2003); Ward et al., J. CLIN. VIR. 29:179-188 (2004). For those assays that have been directed to the INFA H5 and H7 HA subtypes, the results of the assays were not found to be superior over traditional serotyping for HA subtypes. Spackman et al., J. CLIN. MICROBIOL. 40(9):3256-3260 (2002); Munch et al., ARCH. VIROL. 146:87-97 (2001); Lee & Suarez, J. VIROL. METH. 119:151-158 (2004).
In order to be able to detect and treat INFA and IFNB viruses in humans and animals, there remains a need in the art for highly sensitive assays that are capable of detecting all INFA and INFB strains and subtypes.